Cellooligosaccharide is a general term for cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose and is one of oligosaccharides where 1 to 6 glucopyranose units are joined together through β-1,4 linkages.
In recent years, the physiological function of the cellooligosaccharide is being elucidated as with other oligosaccharides. Therefore, the cellooligosaccharide is expected as a novel raw material for functional foods (Non-Patent Document 1).
The cellooligosaccharide is obtained by hydrolyzing its polymer, cellulose, with an enzyme. However, naturally-occurring cellulose is hardly soluble in water and highly crystalline and is therefore less likely to undergo enzymatic decomposition by cellulase. Thus, this has presented a problem.
In the enzymatic decomposition reaction of cellulose, cellooligosaccharide obtained as a decomposition product is further decomposed into glucose units by β-glucosidase that is a component in cellulase, thereby causing reduction in the yield of cellooligosaccharide. Thus, this has presented another problem (Non-Patent Document 2).
In light of the above-described problems, many attempts have heretofore been made for the purpose of improving the yield of cellooligosaccharide at the time when cellulose is enzymatically decomposed.
Processes of producing cellooligosaccharide using particular cellulose include the followings:
Patent Document 1 has described a process of producing cellooligosaccharide, in which a cellulose raw material containing amorphous cellulose in large amounts is used and subjected to hydrolysis reaction with cellulase in the presence of lignin while at least cellobiose of cellooligosaccharides generated by the hydrolysis reaction is collected from the reaction solution at any time.
Patent Document 2 has described a process of producing cellooligosaccharide, in which wet pulp, which is obtained without undergoing drying after the cooking of a natural lignocellulose-containing raw material, is partially hydrolyzed with cellulase to collect at least cellobiose of generated cellooligosaccharides. In these production processes, cellooligosaccharide-decomposing enzyme β-glucosidase contained in cellulase is adsorbed into lignin and inhibited from action of β-glucosidase, thereby reducing the decomposition of the cellooligosaccharide into glucose to enhance the reaction selectivity of cellooligosaccharide. However, in these production processes, the resulting saccharification liquid contains lignin in large amounts, with the result that the yield of cellooligosaccharide is reduced. Moreover, because treatment for eliminating lignin from the saccharification liquid is required for obtaining cellooligosaccharide of high purity, a complicated purification step has been a problem.
Patent Document 3 has described a process of producing cellobiose, a type of cellooligosaccharide, by reacting lignocellulose containing 1 to 20% by mass of lignin with cellulase and a lignin-decomposing fungus such as a white-rot fungus. This process can enhance the action of cellulase on the substrate without treatment for eliminating lignin in cellulose. However, its decomposition product contains not only cellobiose but also lignin decomposition products, thereby causing reduction in the yield of cellooligosaccharide as in the above-described processes. Moreover, because a step of removing the lignin decomposition products is required for obtaining cellobiose of high purity, a complicated purification step has been a problem.
Patent Document 4 has described a process of producing cellooligosaccharide, in which, after cellulase is added to a cellulose solution obtained by dissolving cellulose in solvents such as amine oxide, lithium chloride/N,N-dimethylacetamide, cuprammonium, and viscose, cellulase-containing regenerated cellulose is obtained from the resulting solution and subsequently subjected to enzymatic reaction by the cellulase contained in the regenerated cellulose in the presence of a buffer to produce cellooligosaccharide. The process does not require special pretreatment such as purification given to cellulase and improves the yield of cellooligosaccharide. However, because the process requires a step of dissolving and regenerating cellulose, a complicated step has been a problem. Chemical substances used in the dissolution of cellulose, such as amine oxide, lithium chloride/N,N-dimethylacetamide, cuprammonium, and viscose, have no small action on cellulase. As a result, there has been a problem in that decomposition reaction of cellulose is affected by the chemical substances.
Patent Document 5 has described a process of producing cellobiose using, as a raw material, bleached slush pulp having water retentivity between 230 and 280% and drainability between 550 and 640 ml. The slush pulp used therein is undried pulp after cooking/bleaching treatment. Although the use of the pulp as a raw material certainly improves the production quantity of cellobiose, undried slush pulp is restricted in substrate concentration at the time of enzymatic decomposition due to its high water retentivity. Thus, the poor productivity of cellooligosaccharide has been a problem.
Patent Document 6 has described a process in which, after a cellulose component is solubilized from a cellulose-containing material using supercritical or subcritical water, the resulting treated liquid is supplemented with a cellulase preparation, and cellulose and cellooligosaccharide (partial decomposition product of cellulose) having a high degree of polymerization are hydrolyzed with the cellulase preparation, thereby obtaining glucose and/or cellooligosaccharide. This process improves both of the production quantity and the yield of cellooligosaccharide such as cellobiose and cellotriose. However, there remains an issue surrounding the safety of cellulose pretreatment, for example, the safety of facilities such as pressure-resistant/acid-resistant facilities required for supercritical or subcritical water treatment and the safety against pressurization and heating.
Patent Document 7 has described a process of producing cellooligosaccharide using, as a reaction substrate for cellulase, pulp having a degree of cellulose I crystallization from 10% to 80% determined by X ray diffractometry and water retentivity from 200% to 1000%, wherein the pulp is subjected to any one or several treatment(s) selected from fibrillation treatment, mechanochemical treatment, and chemical treatment. The process improves the production quantity of cellobiose and the decomposition rate of cellulose. However, because of use of fibrous pulp with high water retention as cellulose the process presents such a problem of poor productivity of cellooligosaccharide that clogging occurs, substrate concentration is restricted, and so on at each producing step such as pretreatments (e.g. fibrillation treatment, mechanochemical treatment, and chemical treatment) and, the subsequent enzymatic decomposition and purification of oligosaccharides. The process is essentially different from the process of the present invention in which an average degree of polymerization, an average particle size, a colloidal component content, and so on, are controlled at high level and treating properties at each step including enzymatic decomposition are enhanced.
Methods for improving the yield of cellooligosaccharide by enzymatically decomposing cellulose with particular cellulase include the following Patent Documents 8 to 11:
Patent Document 8 has described a process of producing cellooligosaccharide from a cellulosic material in an aqueous reaction solution by the action of cellulase produced by a microorganism belonging to the genus Cellvibrio, in which an ultrafiltration reactor is used in combination, so that production inhibition is removed to produce and accumulate cellooligosaccharide. According to this process, cellooligosaccharides consisting only of cellobiose and cellotriose are obtained as decomposition products from the enzymatic decomposition of the cellulosic material. However, because an enzyme produced by a microorganism of the genus Cellvibrio is less likely to act on crystalline cellulose, amorphous cellulose as a substrate is required for reducing reaction time and improving yields. Thus, a complicated step has been a problem.
Patent Document 9 has described a process of producing cellooligosaccharide by decomposing cellulose with cellulase, in which cellulase is brought in advance into contact with a weakly acidic cation-exchange resin equilibrated to pH 3.5 to 5.0 to thereby selectively remove β-glucosidase in the cellulase, and the cellulase from which β-glucosidase has been removed is brought into contact with cellulose. According to the process, glucose is reduced by the enzymatic decomposition of the cellulose, so that a decomposition product having 60% or more cellooligosaccharide can be obtained. However, the above-described method requires a step of removing β-glucosidase in cellulase. Thus, there has been a problem in that a step of producing cellooligosaccharide is complicated. Moreover, because this step of purifying cellulase requires the amount of a cation-exchange resin 75 to 1000 times greater than that of untreated cellulase, the amount of cellulase treated is limited and the productivity of cellooligosaccharide is not sufficient. Thus, there has been a problem in that the cost of cellulase purification and the cost of separation/purification agents of the cation-exchange resin are high.
Patent Document 10 has described a process for cellulase purification in which, after cellulase is dissolved along with either or both of cellulose ester or(and) cellulose ether ester and incubated for a fixed period of time, pH is changed and an insolubilized solid fraction is separated from the solution to thereby selectively remove β-glucosidase in the cellulase; and a process for cellobiose production in which cellulose together with the cellulase from which β-glucosidase has been removed is added to an aqueous medium to make a suspension which is in turn incubated for a fixed period of time to produce cellobiose into the suspension, followed by the collection of the cellobiose.
Patent Document 11 has described a process for cellulase purification in which, after chitosan and cellulase are dissolved in an aqueous medium whose pH has been adjusted to pH that renders the chitosan soluble and are incubated for a fixed period of time, pH is changed and an insolubilized solid fraction is separated from the solution to thereby selectively remove β-glucosidase in the cellulase; and a process for cellobiose production in which cellulose together with the cellulase from which β-glucosidase has been removed is added to an aqueous medium to make a suspension which is in turn incubated for a fixed period of time to produce cellobiose into the suspension, followed by the collection of the cellobiose. These processes improve the yield of cellobiose by subjecting cellulase to adsorption/separation treatment with a cellulose derivative or chitosan and bringing cellulose into contact with the cellulase that remains adsorbed in the cellulose derivative or chitosan. However, these processes require treatment for purifying cellulase and therefore complicate a producing step. There has been a problem in that high costs are needed because the cellulose derivative and the chitosan used in the purification of cellulase are expensive. Moreover, since the cellulase is used with the cellulose derivative and the chitosan in the enzymatic decomposition of cellulose, there has also been a problem in that a step of removing them from the decomposition reaction solution is required.
Heretofore, a process has not been known, by which a water-insoluble natural cellulosic material whose average degree of polymerization, average particle size, colloidal cellulose component content, and diethyl ether-soluble substance content are controlled by pretreatment to fall within a certain range is used as a substrate and enzymatically decomposed with cellulase whose activity ratio of β-glucosidase activity to crystalline cellulose-decomposing activity (β-glucosidase activity/crystalline cellulose-decomposing activity) is controlled to fall within a certain range, thereby enhancing the decomposition rate of cellulose in a short time to selectively produce cellooligosaccharide in high yields.    [Non-Patent Document 1] Cellulose Communications, 5, No 2, 91-97 (1998)    [Non-Patent Document 2] “Cellulase” published by Kodansha Scientific, 97-104 (1987)    [Patent Document 1] JP-A-05-317073    [Patent Document 2] JP-A-07-184678    [Patent Document 3] JP-A-08-89274    [Patent Document 4] JP-A-08-308589    [Patent Document 5] JP-A-09-107087    [Patent Document 6] JP-A-2001-95594    [Patent Document 7] JP-A-2005-68140    [Patent Document 8] JP-A-01-256394    [Patent Document 9] JP-A-05-115293    [Patent Document 10] JP-A-05-227957    [Patent Document 11] JP-A-05-227958